Project Summary Argonautes are the only known family of proteins that can be programmed with any RNA or DNA sequence to make sequence-specific regulators of transcription, mRNA stability, or translation. Our goal is to understand the biology and mechanism of paradigmatic examples of Argonaute proteins and pathways, and, ultimately, to use these insights to design and improve small RNA-guided therapies for human diseases. Indeed, studying how Argonautes work and how their small RNA guides are made has led to the development and FDA approval of small RNA drugs. Nevertheless, fundamental questions about the specificity and function of Argonaute protein-mediated pathways remain unanswered. Despite >20 years of study, for example, we still cannot predict how Dicer enzymes will cleave a pre- miRNA based only on its sequence. We will use biochemical and structural approaches to identify the features that determine where Dicer cleaves a pre-miRNA and how Dicer partner proteins alter this process. In animals, the PIWI subfamily of Argonaute proteins uses 23?30-nt ?piRNA? guides to silence transposons or regulate gene expression in germ cells. piRNAs are made from specific long, single-stranded precursor RNAs. Our research seeks to explain why some genomic regions and transcripts are destined to make piRNAs, while others are excluded. By studying piRNAs in flies, moths, and mice, we hope to identify both evolutionarily ancient and newly evolved strategies that animals use to designate piRNA precursors and to convert them into functional complexes with PIWI proteins. While experimental and computational studies have dramatically improved our ability to predict miRNA targets, similar advances have not yet been made for piRNAs. In the spermatocytes of placental mammals, pachytene piRNAs are nearly as abundant as ribosomes, but we still do not know what or how they regulate. Mutations in the proteins that make pachytene piRNAs cause male infertility, suggesting that pachytene piRNAs promote sperm development. We will use biochemistry and mouse genetics to study the function and specificity of pachytene piRNAs. Finally, 30% of bacterial genomes encode Argonautes, yet we do not know what they do. Surprisingly, we find that in Thermus thermophilus, the DNA-guided, DNA-cleaving Argonaute (TtAgo) acts together with gyrase A to ensure successful replication. Our hypothesis is that TtAgo has acquired a role in disentangling the circular chromosomes at the end of DNA replication, perhaps to compensate for the absence of Topoisomerase IV in this organism. We will use genetics and biochemistry to understand how TtAgo acquires its guides, and how and what it regulates in vivo. Together these studies will reveal diverse strategies that organisms use to make small RNAs and how they use Argonautes to control development, differentiation, and reproductive health.